Highly active antiretroviral therapy (HAART) drugs primarily target three of the four HIV enzymes: reverse transcriptase (RT) DNA polymerase, protease, and integrase. Although HAART is very effective in suppressing viral load in HIV-infected patients, prolonged treatment inevitably leads to the emergence of drug-resistant viral strains. Hence, it is essential to develop agents that act on novel HIV targets. The fourth HIV enzyme, RT- associated ribonuclease H (RNH) is one such target. RNH degrades the viral RNA genome during reverse transcription and is essential for HIV replication. It is the only enzymatic activity of HIV that has yet to be addressed by antiretroviral drugs. Such drugs will likely be active against all current drug-resistant viral strains. Our goal is to develop potent RNH inhibitors (RNHIs) with nanomolar efficacy in cell-based replication assays and for this we will use a multidisciplinary approach based on our extensive expertise in structural biology, computational biology, medicinal chemistry, enzymology, biochemistry and virology. We will pursue improvement of current leads and achievement of nM potencies of antiviral activities through a structure-based design process involving iterative cycles of structure determination and computational analysis of RNH-RNHI complexes, medicinal chemistry, and biochemical and virological characterization of newly synthesized inhibitors. To this end we propose three specific aims: Specific Aim 1. Structure-Activity Relationships (SAR) and Chemical Synthesis. We will prepare a database of the validated RNHI screening hits and novel scaffolds that we have developed. We will perform complete SAR for two different classes of RNHIs based on the database analysis and the structural information gained from crystallographic and molecular docking studies in aim 2. Specific Aim 2. Crystallographic and computational analysis of RT-RNHI interaction. Structure-based design is a main focus of this application. We will use crystallographic tools that are already established in our lab and that routinely result in high resolution structures of RT and/or RNH in complex with inhibitors (resolutions up to 1.5 ). The structural information will be used to guide the design of new inhibitors. Specific Aim 3. Biochemical and virologic profiling of RNHIs. We will use biochemical and virological assays to assess selected validated screening hits and new RNHIs to be prepared in Aim 1. This information will be integrated in the iterative SAR-mediated design of new inhibitors. Our multidisciplinary approach will lead to new inhibitors of HIV that will be effective against both wild- type and drug-resistant viral strains.